1. Field of Invention
The present invention provides a method for screening and evaluating a synthetic compound or naturally occurring compound which inhibits an activation of NF-.kappa.B, simply and highly sensitively by utilizing a gene having a sequence which regulates the expression of human inducible nitric oxide synthase (hiNOS) gene.
2. Related Art
It has been clarified that an inflammatory disease is caused by the production of many types of mediators. Development of the remedy of the inflammatory disease is made difficult because the cause of the disease cannot be attributed to one mediator. One of the factors regulating the production of the mediators is activation of NF-.kappa.B.
NF-.kappa.B is a protein which regulates a gene expression, and is one of the so-called transcription factors. When a normal cell is stimulated with an inflammatory cytokine such as interleukin (IL)-1, tumor necrosis factor (TNF)-.alpha., a lipopolysaccharide, UV-rays, or the like, NF-.kappa.B is activated, moved into the nucleus from the cytoplasm, and binds to a specific nucleotide sequence on a genomic DNA, and participates in the expression of various genes (Blackwell, T. S. and Christman, J. W. (1997) Am. J. Respir. Cell. Mol. Biol., 17, 3-9).
Genes which are considered to be subjected to expression regulation by NF-.kappa.B are often those participating in immunity inflammation reactions such as inducible nitric oxide synthase (iNOS), inflammatory cytokines such as TNF-.alpha., IL-1, IL-6 and IL-8, and cell adhesion molecules such as ICAM-1, VCAM-1 and ELAM-1 (Collins, T., Read, M. A., Neish, A. S., Whiteley, M. Z., Thanos, D. and Maniatis, T. (1955) Faseb. J., 9, 899-909). Moreover, when an inflamatory cytokine binds to its receptor, the cytokine is known to transduce a signal which activates NF-.kappa.B through various routes, which is considered to aggravate the inflammation. As explained above, the activation of NF-.kappa.B is understood to be a cause and an exacerbation factor of a variety of diseases (Baeuerle, P. A. and Baichwal, V. R. (1997) Adv. Immunol., 65, 111-137).
Furthermore, it has been reported in recent years that HIV, HTLV-1, CMV, adenovirus, or the like activates NF-.kappa.B in the host cells (Dezube, B. J., Pardee, A. B., Beckett, L. A., Ahlers, C. M., Ecto, L., Allen-Ryan, J., Anisowicz, A., Sager, R. and Crumpacker, C. S. (1992) J. Acquir. Immune Defic. Syndr., 5, 1099-1104, Nabel, G. and Baltimore, D. (1987) Nature, 326, 711-713, Fazley, F., Dezube, B. J., Allen-Ryan, J., Pardee, A. B. and Ruprecht, R. M. (1991) Blood, 77, 1653-1656, Munoz, E. and Israel, A. (1995) Immunobiology, 193, 128-136). Activation of NF-.kappa.B increases the transcription, proliferation and infectivity of the virus.
Furthermore, activation of NF-.kappa.B has been confirmed in various chronic inflammatory diseases (Marok R., Winyard P G, Coumbe A, Kus M L, Gaffney K, Blades S, Mapp P I, Morris C J. Blake D R, Kaltschmidt, Baeuerle P A (1996) Arthritis Rheum. 39, 583-591).
Activation of NF-.kappa.B influences the expression of genes related to a variety of inflammatory diseases, and, as already explained, iNOS is an example wherein the gene expression is influenced thereby. The iNOS is an enzyme whose substrate is L-arginine and which biosynthesizes nitric oxide (NO). For hiNOS, the complete structure of the structural gene including an about 0.4-kb sequence in the 5'-flanking region has been elucidated (Chartrain, N. A., Geller, D. A., Koty, P. P., Sitrin, N. F., Nussler, A. K., Hoffman, E. P., Billiar, T. R., Hutchinson, N. I., and Mudgett, J. S. (1994) J. Biol. Chem., 269, 6765-6772). Excessive NO produced due to the induction of the iNos is thought to wound normal cells to cause various symptoms.
It has been reported that iNOS is induced in any species by inflammation conditions, and it has been shown that the inhibition of the activity or the expression of the enzyme is effective in abatement of the symptom (Cattell, V. and Janse, A. (1995) Histochem. J., 27, 777-784, Nussler, A. K. and Billiar, T. R. (1993) J. Leukoc. Biol., 54, 171-178). Moreover, it has been shown in an experiment of the gene knockout mice of the mouse iNOS(miNOS) that the disruption of the gene leads to resistance to inflammation caused by sepsis or carrageenan. Accordingly, it has become evident that the inhibition of iNOS causes these symptoms (Wei, X. Q., Charles, I. G., Smith, A., Ure, J., Feng, G. J., Huang, F. P., Xu, D., Muller, W., Moncada, S. and Liew, F. Y. (1995) Nature, 375, 408-411).
Accordingly, if activated NF-.kappa.B can be inhibited, it is considered that the expression of not only iNOS but also other genes and viruses that encode inflammatory cytokines and adhesion molecules can be entirely repressed.
Under such situations, a low molecular weight compound is desired which can extensively inhibits the production and expression of protein relating to the cause of inflammation. Such a substance is particularly promising for remedies and prevention against diseases directly or indirectly caused by the activation of NF-.kappa.B, for example, for remedies and preventives against various autoimmune diseases, immunosuppressants, and remedies and preventives against virus infection.
However, since there is no satisfactory evaluation method which can be used for screening substances (specifically, candidate substances for pharmaceuticals) inhibiting the activity of NF-.kappa.B by evaluating the activation of NF-.kappa.B, a simpler and more highly sensitive evaluation method has been desired.